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26
Chapter • Transferase Inhibitors
. Fig. 26.16 Examples of protein ligands that bind to proteins with
atightly bound metal center. Tetracycline 26.33 chelates magnesium
ions so tightly that protein binding of this ligand is achieved together
with the Mg2+ ion. Cisplatin 26.34 binds through substitution of the
. Fig. 26.17 Superposition of the crystal structures of the complex
of PIM-1 kinase with the unselective inhibitor staurosporine 26.21
(light blue) and the selective ruthenium carbonyl complex 26.36 (ol-
ive green). The binding geometry is almost identical in both cases. In
26.36, the carbonyl group is opposite to the β-strand that runs above
the binding pocket. (7 https://sn.pub/JzlPkM)
chlorine atoms by the basic nitrogen atoms of the nucleotide bases of
DNA. Replacement of the sugar moiety in staurosporine 26.21 led
to the chelating ruthenium complex 26.35. They proved to be potent
kinase inhibitors (e.g., 26.36). N-Methylation at the NH function of
26.36 leads to an almost inactive compound (26.38)
The highly similar geometries of the complexes give no
obvious indication as to why the metal center converts
the promiscuous staurosporine scaffold into highly selec-
tive inhibitors. The selectivity prole for other kinases
can be shifted by exchanging the coordinating ligands
on ruthenium and by inverting the stereochemistry. It
remains unclear whether this shift is due to the strongly
altered charge distribution on the scaffold or to the interactions with the polymer chain above the ATP-binding
site. Interestingly, the ruthenium complexes proved to be
active under in vivo conditions, interfering with the signaling cascade of the so-called wnt pathway in human
cell lines and in frog and zebra sh embryos. Time will
tell whether such metal complexes really open up anew
perspective for drug development or whether they serve
as interesting probe molecules for basic research on signaling pathways. Certainly, they will have an answer to
the specic question of developing selective kinase inhibitors, but it remains to be discovered.
26.7 Phosphatases: Reversal Switch
to Activate and Inactivate Proteins
Posttranslational modications of proteins serve to
regulate cellular processes. Phosphorylation by kinases
usually leads to the activation of proteins; the transfer of aphosphate group switches on their biochemi-
. • Phosphatases: Reversal Switch to Activate and Inactivate Proteins
cal function. In order to remove the phosphate group,
which typically leads to the deactivation of abiochemical
function, Nature has developed acounterpart to kinases:
the phosphatases (. Fig.26.1). They can remove phosphate groups from the amino acids Ser, Thr, Tyr, and
His by hydrolysis. There are three families of phospha-
tases. The rst family removes phosphate groups from
serine and threonine. It has two metal ions in its catalytic
site: probably zinc and manganese or magnesium ions
(. Fig.26.18, left). These are held in place by histidine
and aspartic acid residues. Awater molecule (or OH−)
bridges the two metal ions. It is, therefore, highly polar-
ized and can make anucleophilic attack on the phosphate group to be cleaved. The phosphate group also
undergoes polarization and is prepared for nucleophilic
attack by coordinating to the metal ions with two of its
oxygen atoms. The intermediate collapses with the transient formation of apentacoordinated phosphorus atom.
The bond between the hydroxyl oxygen atom of the
Ser or Thr residue and the phosphate group is cleaved.
Aneighboring histidine assists the cleavage by providing
the necessary proton. The reaction is similar to that of
phosphodiesterases (Sect.25.8).
The second group of phosphatases does not use
ametal ion for the cleavage reaction, but acovalent intermediate is formed during the reaction (. Fig.26.18,
right). These phosphatases cleave phosphate groups
from tyrosine residues. The formation of avery deep
binding pocket, about 9 Å long, is characteristic of the
latter phosphatases. It is fully formed only after substrate binding. Aloop containing atryptophan, proline, and aspartic acid (WPD loop) is located above the
catalytic site and closes it to the outside. It contributes
the catalytically important aspartic acid and is critical
for substrate recognition (. Fig.26.18). In the closed
substrate-bound state, aspartic acid forms an H-bond
with the phenolic oxygen atom of the phosphotyrosine
residue. This interaction polarizes the phosphate group
and prepares it for nucleophilic attack. This step is accomplished by an adjacent cysteine residue located near
the end of along helix. In addition, an arginine helps to
stabilize the transition state of the reaction, analogous
to the oxyanion hole in serine or cysteine proteases.
Similar to the acyl–enzyme complex formed in serine
proteases (Sect.23.2), the protein is transiently phosphorylated at the sulfur atom. The dephosphorylated
substrate is released from the catalytic site. In the next
step, awater molecule attacks and cleaves the phosphate
group from the thiol group of the cysteine, which is polarized by the neighboring aspartic acid. This returns
the catalyst to its initial state. The next reaction cycle
can begin.
. Fig. 26.18 Two catalytic mechanisms have been described for the
cleavage of phosphate groups from serine, threonine, and tyrosine in
peptide substrates. The rst group (left) uses two metal ions (presum-
2+
ably Zn
aspartic acid. Awater molecule (presumably in the form of an OH−
group) nucleophilically attacks the phosphate group of the substrate
and initiates the cleavage. The second class of phosphatases begins the
and Mn2+ or Mg2+), which are coordinated by ahistidine or
cleavage reaction with anucleophilic attack by the thiolate group of
acysteine (right). The pK
by the dipole moment of ahelix that is pointing towards the site that
accommodates the thiol group and the reaction starts from a deprotonated cysteine. Finally, awater molecule initiates the cleavage of the
phosphate group from cysteine
value of this cysteine is markedly shifted
a
Chapter • Transferase Inhibitors
26
. Table 26.1 Examples for phosphatases that have been
recognized as target structures for drug therapy
Family Description Disease, therapeutic approach
pSer, pThr PP1, PP2A Tumor suppression
PP2B, PP2C Cystic brosis
(Calcineurin) Immunosuppression
Asthma
Cardiovascular diseases
pTyr PTP-1B Diabetes, obesity
CD45 Alzheimer’s disease
Shp2 Cancer therapy,
immuno-oncology
Dual-specic
phosphatases
VHR, Regulation of MAP kinases
Cdc25
Stimulation of the cell cycle
Anticancer therapy
While the rst and second families of phosphatases
process different substrates by completely different mechanisms, there is athird family that functions similarly to
the second group of tyrosine phosphatases. It has dual
specicity and can cleave phosphate groups from serine, threonine, and tyrosine. Unlike the specic tyrosine
phosphatases, it has ashorter binding pocket that allows
phosphotyrosine as well as the shorter phosphoserine
and phosphothreonine to reach the catalytic site.
So far, the genes for 189 phosphatases have been dis-
covered in our genome. In contrast to protein kinases,
where folding is conserved across all catalytic domains,
phosphatases show greater diversity. So far, 10different
folding patterns have been reported for these proteins.
The majority (106 examples) are phosphatases that use
athiolate group of acysteine residue for nucleophilic
attack. The second largest group with 20examples uses
the two metal ions in the catalytic center. Many phosphatases intervene in signaling cascades by targeted de-
phosphorylation. Most of them remove phosphate groups
from activated proteins, thereby, deactivating the receptors involved. However, the processes can be even more
complicated. Phosphorylation can also hold aprotein
complex in an inactivated state and release its physiological function by removing the phosphate group (see the
example of Shp2 in Sect.26.9). Often, phosphatases are
active as catalytic domains in combination with larger
protein assemblies of signal transduction. Since the
phosphate group as well as the phosphorylated amino
acids and nearby residues are involved in the interaction
with the phosphatase, the selectivity problem is not as
severe as with the kinases. However, the small-molecule
drug to be developed competes with the recognition site
of ahighly polar protein substrate, which does not make
its development any easier. Forty of the 189 phospha
tases (21%) have been identied as targets in many differ-
ent disease areas. Of these, 12are associated with cancer.
For kinases, the number is slightly higher (35%) and the
proportion in cancer is also higher (20%). Some examples
of drug development are summarized in . Table26.1.
The example of PTB-1B, areceptor tyrosine phosphatase
that has been pursued by many pharmaceutical companies as an innovative target enzyme for the treatment of
diabetes and obesity, illustrates how potent inhibitors of
phosphatases can be developed.
26.8 Inhibitors of PTP-1B: Treatment for
Diabetes and Obesity?
Adult-onset type2 diabetes and obesity are diseases that
have increased alarmingly in our society in recent years.
They must be considered as typical diseases of civilization. Adult-onset diabetes is based on increasing insulin
resistance, which is observed as areduced ability of cells
in the target organ to respond to insulin. As aresult, high
blood insulin levels occur even when blood glucose levels
are normal. Because of the resistance, the cells no longer
respond as they should to the signal that insulin would
send in ahealthy person. Insulin causes the uptake of
glucose from food into liver cells, where glucose is stored
in the form of glycogen. As resistance increases, pathophysiological changes occur due to inadequate insulin
control. The uptake of blood glucose into tissues and
the release of glucose from the liver become imbalanced.
As aresult, blood glucose levels rise even higher, which
can lead to complications such as coronary heart disease,
retinopathy, cataracts, and vascular disease.
The other disease of civilization is much more obviously seen: obesity. The signs are adisproportionate
excess of body mass. Even more alarming is the fact that
obesity is by no means limited to old age. Even among
young people, the number of cases of obesity is increasing dramatically. Today, about aquarter of adults worldwide are overweight. In developed countries, the numbers
are much higher. In the U.S., nearly 75% are considered
overweight and 40% are obese. In developing countries,
too, the percentage is rising sharply. Of course, this has
something to do with our changing lifestyles. An overabundance of food, often without dietary ber, coupled
with alifestyle that requires less and less physical labor
has led to this development. In addition, genetic predisposition contributes to the development of obesity.
Interestingly, the development of type 2 diabetes
and obesity often occur together, increasing the health
risks for the patient. The resulting symptoms are called
metabolic syndrome. For this diagnosis, the following
additional criteria apply: an abdominal girth of more
than 80 cm in awoman or 90 cm in aman, and two of
the following additional factors: an elevated triglycer-
-
ide level (> 150 mg/dL), an elevated fasting glucose level
(> 100 mg/dL), arterial hypertension (> 130/85 mmHg),
. • Inhibitors of PTP-B: Treatment for Diabetes and Obesity?
and/or areduced HDL cholesterol level (< 40–50 mg/dL;
Sect.27.3). The cost to society of this increased health
risk is difcult to estimate, but it is likely to be dramatic.
Therefore, great efforts have been made to nd drug therapies that can counteract the metabolic syndrome and its
consequences.
The correlation between insulin resistance and obesity is not yet fully understood at the molecular level. In
fact, insulin is ahormone that is related to fat metabolism
and inuences fat deposition. For example, it inuences
fat storage, but insulin deciency leads to weight loss. In-
sulin is bound to the insulin receptor, which is autophos-
phorylated by its tyrosine kinase domain in response to
this signal (Sect.29.8). This initiates acascade of several
kinases that culminates in the synthesis of the sugar-storing glycogen. The synthesis of fatty acids and proteins
is also induced. Dephosphorylation of the insulin recep-
tor attenuates its function. PTB-1B tyrosine phosphatase
cleaves phosphate groups from two tyrosine residues on
the receptor. This leads to deactivation of the insulin receptor and the cascade initiated by the receptor. Blocking
this dephosphorylation step seems to be arewarding concept to counteract insulin resistance. The real stimulus
for the search for PTP-1B inhibitors was the observation
that mice with aknocked-out ptp-1b gene are resistant to
developing obesity despite no changes to their diet, and
their insulin sensitivity is increased without any apparent negative consequences. This spectacular observation
suggested that the ideal target had been found to ght the
most prominent disease of civilization. This optimism
was reinforced by the fact that antisense nucleotides
(Sect.32.4), which block the expression of PTP-1B, also
cause an increased insulin effect. As aresult, nearly every
pharmaceutical company of note ocked to this enzyme
to develop potent inhibitors. Within four years, more
than 200 patent applications appeared in the literature!
Has PTP-1B proven to be an easy target? The mechanism of action is shown in the previous Sect.26.7. The
catalytic cysteine, which temporarily accommodates
the cleaved phosphate group, aligns itself at the tip of
along helix oriented towards the catalytic site. Such ahelix creates special electrostatic conditions at its terminal end (Sects.30.2 and30.8) and can stabilize charged
species well. The catalytic center also contains an aspartic acid and an arginine. The structure with the phosphorylated tyrosine 26.39 (green; . Fig.26.19) is part
of asubstrate. The complex with this substrate could
be determined because the enzyme was rendered almost
catalytically inactive by replacing the catalytic Cys 215
with an analogous serine, but remained geometrically unchanged. The phosphate group is bound in atight network of H-bonds. The phenyl ring of tyrosine is held in
ahydrophobic clamp by two adjacent aromatic residues,
Tyr46 and Phe 182. These two residues also determine
the depth and width of the entrance to the catalytic site
of the phosphatase (. Fig.26.20, upper left). First, an
. Fig. 26.19 The crystallographically determined binding mode of
aphosphorylated tyrosine (26.39, green, . Fig.26.21) as aminimal
mimetic for apeptide substrate in the human phosphatase PTP-1B.
The phosphate group is held in place by Arg 221 and Cys 225 which
is positioned for nucleophilic attack. Asp 181 is found above the Cys
residue and buffers for the protonation inventory. The entrance to
the binding pocket is bordered by the two aromatic residues Phe
182 and Tyr46. The binding position of the cysteine is found at the
end of along helix. The displayed geometry is based on acrystal
structure with the catalytically inactive Cys →Ser mutant. Asecond
phosphotyrosine (pink) is found in the crystal structure that binds
to Arg24 and Arg 254 in a second distal pocket. Consequentially,
the occupancy of this second binding pocket was important for the
development of nanomolar PTP-1B inhibitors (cf. . Fig. 26.21).
(7 https://sn.pub/QLME5k)
attempt was made to replace the phenolic oxygen atom
of the tyrosine residue attaching the phosphate group
of the substrate 26.39 with anonhydrolyzable mimetic
such as 26.40 (. Fig.26.21). ACF2 group was chosen
to replace the oxygen atom. However, attempts have also
been made to replace the uorine on the bridging carbon
with an OH group. Alternatively, dicarboxylic acids were
considered as head groups. The polar properties of the
compound were essentially retained, but the hydrolytic
stability was signicantly improved. Afragment-based
screening approach using crystallography and NMR
spectroscopy (Sects.7.8 and7.9) was used to discover
oxalic anilide 26.41 and N-oxalylanthranilic acid 26.42
as potential phosphotyrosine mimics. The thiophene
analog 26.43 proved to be asubmicromolar inhibitor.
Surprisingly, in the crystal structure with phosphotyrosine, asecond molecule of 26.39 (pink) was found to be
bound (. Figs.26.19, 26.20, upper left). It binds adjacent to the rst molecule (green) and occupies asecond
pocket formed by Arg24, Arg 254, Gln 262, and Asp48.
However, the afnity for this binding site was only in the
millimolar range. Nevertheless, the discovery led to the
26
ab
cd
Chapter • Transferase Inhibitors
. Fig. 26.20 Upper left Binding mode of the substrate-analogous
phosphotyrosine (26.39, . Fig. 26.21) in human PTP-1B. The phosphate group binds deeply in the catalytic center (green). The two hydrophobic amino acids Phe 182 and Tyr46 form anarrow entry portal
to the catalytic site. Asecond phosphotyrosine (pink) is found in the
crystal structure that binds to Arg24 and Arg 254. Upper right Crystal
structure of an aromatic oxalic acid derivative (26.45) that was developed at Abbott to occupy the catalytic site (green). The compound
induces arearrangement of the Phe 182 side chain and opens the catalytic site to the top. Bottom left By chemically coupling an aromatic
carboxylic acid that was discovered with the SAR-by-NMR method
as abinder for the second binding site (pink) and amimetic to occupy the catalytic site, a nanomolar inhibitor 26.49 (. Fig.26.21) was
obtained. Bottom right To achieve selective binding to PTP-1B com-
pivotal idea of coupling the active site phosphotyrosine
mimetic to amolecular building block occupying this
second binding site. The plan was to create inhibitors
with amuch higher binding afnity.
Aromatic oxalic acid derivatives such as 26.44 and
26.45 have also been worked on at Abbott as substrate
mimics for binding to the catalytic site. Interestingly, the
derivatives pursued by Abbott forced aconformational
change at Phe 182 at the entrance, so that the top of the
catalytic site is opened (. Fig.26.20, upper right). Abbott additionally applied their SAR-by-NMR technique
(Sect.7.8) to discover potential binders for the second
pared to the structurally very similar TCPTP, structural differences
at position41 were exploited (light blue). There PBP-1B has alysine,
and the related family member TCPTP has an Arg in this position.
The nanomolar inhibitor 26.53 (green) achieves asignicant selectivity
advantage. 26.52 (light blue) binds with an uncharged head group into
the catalytic center. The nanomolar inhibitor 26.51 (purple) also binds
into the catalytic center but, like 24.49, its oriented towards the second
phosphotyrosine binding site (pink). (7 https://sn.pub/D1DtFZ)
binding site. Small aromatic acids such as 26.46–26.48
were discovered. By coupling such moieties (e.g., naphthyl
carboxylic acids) and the already known mimetic 26.45
to bind to the catalytic center produced the nanomolar
inhibitor 26.49 (Ki = 22 nM, . Fig.26.20, lower left).
This second binding site was determinant for the
lead structure optimization. At Novo Nordisk, the initial oxalic acid derivatives on the thiophene ring were
expanded by using Asp48 as an additional anchor point
to arrive at more potent and selective inhibitors based on
scaffold 26.50. Wyeth also focused more on the second
binding site and developed adicarboxylic acid derivative
. • Inhibitors of PTP-B: Treatment for Diabetes and Obesity?
. Fig. 26.21 By starting with asubstrate with aterminal phosphoty-
rosine 26.39, ahydrolytically stable compound 26.40 was developed.
Afragment screening drew attention to the two mimetics 26.41 and
26.42. Thiophene derivatives such as 26.43 were designed from the
latter compound. At Abbott, analogous aromatic oxalic acid derivatives 26.44 and 26.45 were developed. Screening by the SAR-by-NMR
method discovered aromatic carboxylic acids such as 26.46–26.48 as
ligands for the second binding site. By chemically linking such aromatic carboxylic acids as binders for the second binding site and amimetic
on athiophene ring 26.51 as asingle-digit nanomolar
inhibitor. Also of note is compound 26.52 from Incyte
Corporation, which binds to the catalytic center with an
uncharged head group (. Fig.26.20, lower right).
The development of highly potent, PTP-1B selective,
and orally available inhibitors was overshadowed by another observation. Sequence comparisons suggested that
there is another phosphatase, the T-cell protein tyrosine
phosphatase TCPTP, which is highly similar to PTP-1B.
Such an observation is worrisome because the PTP-1B
inhibitors in development may also inhibit this phosphatase. The crystal structure published in 2002 conrmed
this suspicion: the sequence identity of the catalytic domains is 74%, and the WPD loop, which is located above
the catalytic site after substrate binding, is identical.
Knock-out mice lacking the tcptp gene are born healthy
but die within 3–5weeks of birth. More alarmingly,
knocking out both the ptp-1b and tcptp genes simulta-
for the phosphotyrosine in the catalytic site, 26.49 was obtained as
ananomolar inhibitor. Also at Novo Nordisk, the rst lead structures
were equipped with side chains for the second binding site (26.50).
The thiophene derivative 26.51 orients from the catalytic center to the
second binding site and achieves nanomolar afnity. Inhibitor 25.52
binds to the catalytic center with an uncharged head group. With
26.53, afourfold more selective inhibitor of PTP-1B than TCPTP was
prepared
neously resulted in animals that had no chance of survival. This underscores the extreme danger that insufciently selective PTP-1B inhibitors that also inhibit T-cell
protein tyrosine kinase could lead to alife-threatening
situation. The need was great. What are the structural
differences between the two phosphatases that could be
exploited to design sufciently selective compounds? All
of the inhibitors developed at that time showed almost
equipotent afnity for both proteins. Bidentate inhibitors
such as 26.53 (. Fig.26.21), reported in 2003, proved
very interesting because they occupy the catalytic site
and neglect the second binding site (. Fig.26.20, lower
right). Even the sequence of this region turned out to
be virtually identical to that of TCPTP. With aslightly
different orientation, the new inhibitors target alysine
residue (Lys41), which is an arginine in TCPTP. At least
the nanomolar inhibitor 26.53 has amodest selectivity
advantage for PTP-1B compared to TCPTP.
26
Chapter • Transferase Inhibitors
The Sunesis company took acompletely different
approach. In 2004, they reported the discovery of an
allosteric binding site 20 Å away on the back side of the
catalytic site in PTP-1B. An inhibitor that binds with micromolar afnity to the enzyme was developed for this
site. It blocks its function by preventing the closure of
the WPD loop. In this way, the loop cannot fold upon
the substrate-binding site. The essential residues such
as the catalytically active aspartic acid are not brought
in the vicinity of the substrate. The most potent ligand
from this series, 26.54 (IC50 = 8 μM), wraps itself around
aphenylalanine that is found there, as proven by the crystal structure (. Fig.26.22). In the structurally analogous
TCPTP, acysteine is found at this position and forms entirely different interactions with the aromatic groups of
this ligand. Due to the deviating interaction pattern, this
compound achieves TCPTP inhibition at only 280 μM.
Perhaps blocking this allosteric binding site will open
anew perspective for the selective inhibition of PTP-1B.
The future must show whether the severe selectivity problem can be resolved in an appropriate way. It should be
noted that all hopes for inuencing this seemingly ideal
target protein are currently focused on the antisense nu-
cleotide mentioned above, which is currently in clinical
trials (Sect.32.4).
. Fig. 26.22 A new allosteric binding site was discovered at Sunesis
that is approximately 20 Å away from the catalytic site of the phosphatase. Compound 26.54 inhibits PTP-1B 16-fold more strongly than
TCPTP. The crystal structure with PTP-1B shows that the inhibitor
basically wraps itself around the exposed Phe 280. In TCPTP, acysteine residue is found in the same position. (7 https://sn.pub/92STnr)
26.9 Molecular Glue Inhibits the Release
of Phosphatase Activity
Focusing on allosteric binding sites seems to be amuch
more promising approach for the development of phosphatase inhibitors than blocking the very polar catalytic
center. It is inevitable that inhibitors of this site will be
very polar. Therefore, they will unavoidably have bioavailability problems. As mentioned above, phosphatases
are often involved as catalytic domains in larger protein
assemblies for signal transduction. In terms of drug
therapy, it is important whether or not such acomplex
is modulated in its biological function. Modulation can
also be achieved by drugs that act on these complexes
at acompletely different site. Thus, we are not limited
to blocking the active site of the phosphatase domain.
Asuccessful example of this approach is the development of the Shp2 inhibitor by Novartis. We will see that
in this example, dephosphorylation removes autoinhibition and consequently releases ablocked phosphatase
function. Therefore, maintaining autoinhibition is apromising strategy to indirectly inhibit phosphatase activity.
Shp2 is aphosphatase belonging to the nonreceptor
tyrosine phosphatase subfamily. It is responsible for reg-
ulating numerous signaling pathways in normal and glioma cells. As aresult, it is an anticancer target and plays
an important role in immuno-oncology. Inhibition of
Shp2 phosphatase has emerged as apromising approach
for drug development against glioblastoma multiforme
(amalignant brain tumor), adreaded cancer with poor
prognosis and low survival. As seen with PTP-1B, the
high positive charge near the active site poses signicant
problems for inhibitor development, particularly with
respect to sufcient cell permeability and bioavailability.
While numerous small-molecule inhibitors of Shp2 have
been described, their polar nature means that they are
simply not ideal for therapeutic development.
Shp2 phosphatase consists of a catalytic phosphatase domain and two SH2 domains (. Fig.26.23). In
the absence of aphosphorylated tyrosine substrate, the
N-terminal SH2 domain binds directly to the phosphatase
domain and blocks its active site. Aloop of the N-SH2
domain inserts itself into the catalytic center. Like aconformational switch, it either inhibits the phosphatase or
binds phosphoproteins and activates the enzyme.
The Novartis researchers started with the concept of
nding an allosteric inhibitor that would block the activation of the phosphatase function. They rst performed
ahigh-throughput screen using the entire Shp2 protein.
Since inhibitors of the catalytic center were undesirable
and should be discarded, the screening was repeated
with the hits found, but now only with the truncated
. • Inhibitors of Catechol-O-Methyltransferase
. Fig. 26.23 Left When no phosphorylated substrate is present, the
Shp2 phosphatase is self-inhibited by its N-SH2 domain. For this purpose, this domain binds with an exposed loop (red box, yellow loop)
via Asp61 to the catalytic center of the phosphatase domain (Cys 459,
purple). Right Only in the presence of a phosphorylated substrate is
the phosphatase domain exposed and can become catalytically active
phosphatase domain without the SH2 domains. This revealed among the nondiscarted hits from the rst screen
the aminopyrimidine 26.55 as apromising candidate. In
the crystal structure with full-length Shp2, this hit was
found to be an allosteric binder that interacts with all
three domains of Shp2 (. Fig.26.24). Thus, it keeps
Shp2 in the autoinhibited, inactive conformation. The
aminopyrimidine 26.55 was optimized through several
design cycles to the pyrazine 26.56, aselective, well soluble, orally bioavailable, and potent Shp2 inhibitor. It has
shown promising antitumor activity in animal models.
The compound is now being tested in more advanced
clinical trials. This success story shows that there are alternative ways to block the function of aseemingly “undruggable” phosphatase with asmall molecule inhibitor.
If necessary, this can also be done in an indirect way!
(based on agure in Fortanet etal., J.Med. Chem., 59, 7773–7782
(2016)). (7 https://sn.pub/lEIYDj)
26.10 Inhibitors of Catechol-O-
Methyltransferase
Alarge family of transfer enzymes are the methyltransferases, which add methyl groups to other biomolecules.
DNA methyltransferases are an important group in
this family. Their function is to chemically modify nucleobases at specic sites on DNA or RNA by transferring methyl groups. These methylations do not alter
the genetic code, meaning the same amino acids are still
translated into the gene product. However, they serve as
akind of label for DNA strands, e.g., to distinguish between the cell’s own and foreign DNA or to distinguish
between original and newly synthesized strands. Another
group of methyltransferases transfer methyl groups to
26
Chapter • Transferase Inhibitors
. Fig. 26.24 Crystal structure of full-length Shp2 phosphatase in
the inactive form autoinhibited by the N-SH2 domain. The compound
26.55 discovered in ascreening campaign could be optimized to the
potent inhibitor 26.56, which, like amolecular glue, stabilizes the inactive form of Shp2 by simultaneously binding to the three domains
(yellow box). (7 https://sn.pub/VfL9Ak)
oxygen, nitrogen, or sulfur atoms in small biomolecules.
Methyltransferases use S-adenosyl-l-methionine (SAM
26.57) as acofactor (. Fig.26.25). In the transmethyl-
ation reaction, ahighly reactive methyl group is transferred from the sulfonium group of this donor molecule
to the substrate.
Inhibitors of catechol-O-methyltransferase (COMT)
have gained importance in pharmaceutical therapy. This
enzyme deactivates the endogenous function of catecholamines such as dopamine, adrenaline, or noradrenaline
by transferring amethyl group to the phenolic hydroxyl
group of these neurotransmitters. Polymorphisms in this
enzyme have been associated with psychiatric changes that
may be related to anxiety disorders and schizophrenia.
Inhibitors of this enzyme are used in therapy, particularly
in the treatment of Parkinson’s disease. This disease, originally known as “shaking palsy,” occurs primarily in older
people. It is caused by aslow, progressive degeneration of
dopaminergic neurons in the substantia nigra of the mid-
brain. Acausal treatment of the neuronal degeneration
has not yet been achieved. Therefore, attempts are being
made to counteract the dopamine deciency with exogenous replacement substances. The amino acid l-DOPA
has already been introduced in Sect.9.4 as aprecursor of
dopamine. Although it has amore polar character than
dopamine, it can penetrate the blood–brain barrier because it uses an amino acid transporter to enter the brain.
In practice, however, only about 1% of the administered
. • Inhibitors of Catechol-O-Methyltransferase
. Fig. 26.25 The crystal structure of COMT with the cofactor
S-adenosyl-l-methionine 26.57 (magenta carbon atoms) and the catecholamine-analogous nitro-substituted inhibitor 26.58 (green carbon
atoms). The methyl group that is to be transferred to the phenolic
oxygen atom (red) is within ashort distance (2.63 Å, violet line). The
phenolic oxygen, which is the nucleophile in the transfer reaction, is
presumably deprotonated because of the electron-withdrawing effect
of the nitro groups and the close proximity to the magnesium ion, the
sulfonium group, and the ammonium group of Lys 144. The accumu-
amount reaches the brain. The vast majority is degraded
in the periphery by decarboxylases. To prevent this degradation and the side effects associated with peripheral dopamine release, adecarboxylase inhibitor is administered
at the same time. This inhibitor must be sufciently polar
to prevent it from crossing the blood–brain barrier (e.g.,
benserazide 9.39, . Fig.9.9). This strategy signicantly
increases the bioavailability of l-DOPA in the brain. The
drug is degraded by monoamine oxidases (Sect.27.8) and
by catechol-O-methyltransferases. COMT recognizes both
l
-DOPA and dopamine as substrates. They are inactivated
lated positive charges also shift the pKa value of this hydroxyl group
into the acidic range. The second phenolic OH group is probably uncharged and forms an H-bond to Glu 199. (7 https://sn.pub/QTslOa)
by the transfer of a methyl group to their phenolic hydroxyl groups. Inhibition of COMT allows the bioavailability of l-DOPA to be further enhanced and a higher
concentration of dopamine to be achieved in the brain.
The crystal structure of the enzyme was solved in 1994
by the group of Anders Liljas at Lund University, Sweden
(. Fig.26.25). The mechanism involves adeeply buried
magnesium ion that assumes an octahedral coordination
geometry. The adjacent oxygen atoms of the catecholamine are chelated with the magnesium ion. This brings
the phenolic oxygen atom into close proximity (2.63 Å) to